We developed systems to investigate hepatocyte lineage life history dynamics in vivo. We propose to define the factors that determine hepatocyte lineage birth-rates and longevities, and to describe their dynamic responses to hepatic stresses in aging. In this collaborative proposal, empirical in vivo studies are combined with mathematical modeling and simulation to test effects of extrinsic and intrinsic factors on hepatocellular lineage dynamics and how these change as a part of the aging process in a genetically tractable animal model. What is known. Unlike most differentiated cell types, hepatocytes can proliferate. When normal liver cells are transplanted into mice having a genetic defect that autonomously compromises the endogenous hepatocytes, the grafted cells can complete >18 consecutive replicative cycles, resulting in full replacement of the endogenous hepatocytes and reconstitution of the liver with healthy cells. Using serial reconstitution through 7 consecutive recipient mice, a classic study showed that adult wild-type liver cells could undergo an average of at least 69 consecutive divisions 1. Thus, liver cells have a nearly unlimited capacity to proliferate 1 and may be stem cell-like in their regenerative immortality 1,2. Hepatocytes are also one of few cell types that undergo endoreplication and acytokinetic mitosis, resulting in polyploid nuclei and bi-nucleate cells, respectively. Most hepatocytes are polyploid and both diploid and polyploid hepatocytes can proliferate. Indeed, another study showed that the most active liver cell types for reconstituting compromised liver are polyploid hepatocytes 3. These background data indicate that: (1) liver cell populations may be infinitely proliferative; (2) hepatocytes are predisposed to becoming polyploid; and (3) polyploid hepatocytes are highly proliferative. Preliminary observations and the problem they reveal. We developed genetic marker systems for time-stamping hepatocyte lineages in vivo 4. In contrast to the prevailing model of the immortal hepatocyte, our systems show that hepatocyte lineages have both a finite half-life and a limited capacity to proliferate. We also developed a flow cytometry-based method of quantifying liver nuclei on the basis of ploidy and found that adult livers between 2- and 12-months of age exhibited nearly invariant ratios of diploid (2N), 4N, and 8N nuclei. Lastly, we developed a novel ten-day chronometer for newly differentiated hepatocyte lineages that allows us to quantitatively assess the contributions of pre-hepatocytic stem cells to liver growth, regeneration, and maintenance 5. Using this chronometer, we found that normal adult liver is continuously gaining new diploid hepatocyte lineages. We believe these replace lineages that die-off due to age or stress. Based on our observations, we suspect that only pre-hepatocyte cell types, not differentiated hepatocytes, have unlimited proliferative potential and that this rare population of cells underlies the proliferative immortality of liver. Our hypothesis and how we will test it. Based on our findings, we hypothesize that hepatocytes have a life history that includes birth from stem cells, age-related deterioration, and death. We predict that hepatic stresses, replication, ploidy, aneuploidy, and time will affect hepatocyte lineage life history dynamics. Moreover, the process of aging of the host animal might influence the life history dynamics of hepatocyte lineages. The quality of either the hepatic stem cells or the liver niche could change as livers age, resulting in differences in hepatocyte lineage birth rates, longevities, proliferative potentials, and stress resistance. To test our hypothesis, we will fulfill four aims: (1) Define birth-rates and longevities of hepatocyte lineages under normal and stressed conditions. (2) Determine what factors limit lineage longevity. (3) Measure the dynamic lineage-aging process in hepatocyte nuclei. (4) Examine how an animal's age and exposure-history affects the life history dynamics of hepatocyte lineages. Implications for human health in aging. Hepatocytes are generally thought to have a stem cell-like capacity to proliferate and regenerate lost or damaged liver tissue. Indeed, the term 'stem cell-like' invokes a level of immortality that has been tested in only a small number of situations. Clearly mouse ES cells, for example those we used to make our various lines of mice having targeted mutations, have been verified through years of culture and mouse-production as being indefinitely self-renewing; but is this true of all organ-specific stem cells? Maintenance of the self-renewing capacity of ES cells in culture requires a very strict environment or niche (e.g., media, supplements, attachment factors, feeder-cells, pH, etc.), so it may be reasonable to predict that the quality of organ-specific stem cells in vivo could also be intimately dependent on the niche that the host-organ provides. This niche could change with an animal's age or exposure history, yet these possibilities have not been previously considered. Here we propose an investigation into the aging process in differentiated hepatocyte lineages and how both this process and the contributions of hepatic stem cells change as animals age. Upon completion of this project, we will have: (a) developed and publicly disseminated novel mouse models for studying aging of hepatocyte lineages; (b) defined rates of birth and death of hepatocyte lineages under normal and several stressed states; (c) characterized the aging process in normal and stressed hepatocyte lineages; and (d) investigated how these processes change as a function of aging and exposure history of the host animal.